The overarching goal of this proposal is to establish the mechanisms responsible for proper segregation of chromosomes during cell division. High vs. low fidelity of chromosome segregation is a characteristic difference between normal vs. cancer cells and understanding the mechanisms responsible for this difference is an essential step towards developing novel anti-cancer therapeutics and treatments strategies. For proper segregation, each chromosome must attach to the microtubules of mitotic apparatus (termed the 'spindle') and this attachment is mediated by special macromolecular assemblies on the chromosome's body known as the 'kinetochores'. The project is enabled by recent demonstrations that geometric constraints of microtubule/kinetochore interactions play a vital role in ensuring proper formation of the mitotic spindle essential for faithful chromosome segregation. These geometric constraints are established by the distribution and orientation of molecular complexes within the kinetochore and this project is designed to reveal how the architecture of the kinetochore, and the orientation of individual molecular complexes within that architecture, adapt to rapidly changing conditions within the mitotic spindle. We aim to dissect the nature of structural reorganizations known to occur within the kinetochore during different stages of mitosis and different physiological states of the kinetochore. To achieve these goals we use a unique approach in which high- resolution electron-microscopy analyses are conducted on the kinetochores whose behavior and movements were followed in live cells up to the moment of fixation. This approach, termed correlative LM/EM allows us evaluate the structure of kinetochores that are in known functional state thus revealing the exact structural changes that occur during transitions between various physiological states. We will also perform comparative structural analyses of kinetochores upon two functionally-characterized experimental conditions: 1) treatment with low concentration of taxol, a condition known to allow satisfaction of the mitotic checkpoint despite the lack of centromere tension vs. 2) treatment with low concentration of nocodazole, a condition known to prevent satisfaction of the SAC despite highly-stretched centromeres. Together, these studies will allow us to create an accurate model for the structure of the kinetochore and determine how the architecture and the distribution of molecular components within the kinetochore adapt in response to various types of microtubule attachment and various types of chromosome movements.